Throat Swab Culture Positivity and Antibiotic Resistance Profiles in Children 2–5 Years of Age Suspected of Bacterial Tonsillitis at Hargeisa Group of Hospitals, Somaliland: A Cross-Sectional Study

Introduction Tonsillitis is the third most frequently diagnosed infection in the pediatric age and is associated with significant morbidity and loss of school attendance. Throat swab cultures are useful for the confirmation of children with a clinically suspected tonsillitis. However, Somaliland is one of the underdeveloped countries with a low standard of sanitation and poor health seeking culture. Treatment of tonsillitis with antibiotics is irrational and not empirical. This study determined the bacterial throat swab culture positivity and antibiotic resistance profiles of the bacterial isolates among children 2–5 years of age with suspicion of tonsillitis at Hargeisa Group of Hospital, Somaliland. Materials and Methods A cross-sectional study was conducted from March to July 2020. A total of 374 children from 2 to 5 years of age suspicion of tonsillitis was included using a convenient sampling method. Throat swabs were collected, and bacterial isolation and identification were done using standard bacteriological procedures. Antimicrobial susceptibility testing was done using the disk diffusion method. Data on demographic variables and clinical profiles were collected using structured questionnaires. Logistic regression analysis was computed to identify factors associated with bacterial tonsillitis. Results Overall, 120 (32.1%) (95% CI 27.4–36.8%) of children were positive for bacterial throat cultures. Of these, 23 (19.2%) were mixed bacterial isolates. The most frequent bacterial isolates were beta-hemolytic streptococci 78 (55%), Staphylococcus aureus 42 (29%), and Streptococcus pneumoniae 10 (7%). Isolates revealed 83.3–100% rate of resistance to ampicillin. Beta-hemolytic streptococci isolates were 94.9% resistant to ampicillin. S. aureus was resistant to clarithromycin (38%) while S. pneumoniae isolates were 100% resistant to ampicillin. History of tonsillitis (AOR = 0.12; 95% CI = 0.06–0.21), difficulty in swallowing (AOR = 6.99; 95% CI = 3.56–13.73), and attending schools (AOR = 2.98; 95% CI = 1.64–5.42) were found to be associated with positive throat culture. Conclusions Resistance to ampicillin and MDR among beta-hemolytic streptococci and other isolates of throat colonizers in children with clinically suspected of bacterial tonsillitis are major concerns in Hargeisa, Somaliland. Therefore, treatments of cases are recommended to be guided by regular culture and antimicrobial susceptibility testing to prevent complications of tonsillitis and associated antibiotic resistance.


Introduction
Tonsillitis, the infammation of the tonsils, is a contagious disease that can spread through close contact with infected persons, sharing food, drinks, and utensils. Tonsillitis impacts the health of children, the quality of life, causes signifcant morbidity, and loss of time for schooling [1]. Poor living conditions, exposure to environmental pollutants, and indoor air pollution are frequently reported factors associated with tonsillitis among children 2-5 years of age [2].
Troat swabs are neither specifc nor sensitive to microbacteria causing sore throat symptoms; however, current guidelines suggest they are still useful in some cases and the clinical diagnosis of symptomatic patients' needs confrmation by throat culture and the microbiological evidence of bacteria in the throat swab proves the existence of bacteria in the swab site [4].
Te emergence of drug-resistant bacteria in tonsillitis is getting higher every year. Troat colonization with methicillin resistant S. aureus (MRSA) is frequent in children. Irrational use of antibiotics by humans, production of beta-lactamase enzymes, and the formation of bioflms by pathogens are the main reasons for the emergence of drug resistance [5]. Te spread of drug-resistant bacteria has led to treatment failure and the recurrence of tonsillitis among children with poor sanitation and hygiene in underdeveloped countries. Te situation is critical in Somaliland, where antimicrobials are vastly and frequently used irrationally [6]. Tis might increase the emergence of resistance to commonly used antibiotics for the treatment of tonsillitis.
Like other similar health settings in Somaliland, routine culture and antibiotic susceptibility tests are not usually performed as essential parts of patient care in Hargeisa Group of hospitals and treatments are mostly empirical. Published data on throat culture and antimicrobial resistance profles of bacteria, as well as the associated factors in children suspicion of tonsillitis, are limited in Somaliland. Pathogen occurrence and susceptibility profles show substantial geographic variations, as well as signifcant diferences in various populations and environments [7,8]. Asymptomatic children can be the sources of dissemination of bacteria causing tonsillitis to noninfected children at home or at school settings [5]. Tese can lead to a wide range of tonsillar infections. Tus, knowledge of the local bacterial isolate and susceptibility profles is required to detect on time any changes that might have occurred so that appropriate recommendations for optimal empirical therapy of bacterial infections can be made.
Terefore, we present the frst report of the profles of bacterial throat culture isolates and antibiotic resistance and associated factors in children 2-5 years of age with suspicion of bacterial tonsillitis at Hargeisa Group of Hospital, Somaliland.

Study Design, Period, and Setting.
A hospital-basedcrosssectional study was conducted between March and July 2020 in Hargeisa Group of Hospital (HGH), Somaliland. Hargeisa Group of Hospital is located in Maroodi Jeex Region, the capital city of Somaliland known as Hargeisa. According to the 2019 census report from the Central Statistics Department of Somaliland, Hargeisa has a total population of 1.2 million. Hargeisa Group of Hospital is the largest referral public hospital with more than 200 healthcare professionals. It is one of the health hubs in Somaliland. Daily, 50 outpatients and 1 to 4 hospitalized children attend the Pediatric Clinic for diferent medical conditions. Children with tonsillitis are diagnosed clinically and managed without the guidance of culture and antimicrobial susceptibility testing. All children aged 2-5 years suspicion of tonsillitis at ear, nose, and throat (ENT) of HGH were the study population.

Inclusion and Exclusion
Criteria. Children 2-5 years of age with either sore throat or swollen tonsils, difculty swallowing, white pus-flled spots on the tonsils, or swollen lymph nodes with or without fever (temperature >38°C at presentation) were considered symptomatic for bacterial tonsillitis [4]. On the other hand, children who were on antibiotics 2 weeks prior to recruitment or had tonsillectomy were excluded from the study.

Sample Size and Sampling
Procedure. Te sample size was calculated using the single population formula n � (Zα/ 2) 2 P (1 − P)/d 2 , where n � sample size, Z � level of confdence according to the standard normal distribution, P � sample proportion, and d � tolerated margin of error. Terefore, by taking Z (α/2) � 1.96 for a level of confdence of 95%, P � 0.5, which is the maximum proportion of positive throat cultures and 5% margin of error, the sample size was calculated as n � (1.96) 2 × 0.5 (1 − 0.5)/(0.05) 2 � 384. All children 2-5 years of age suspected of bacterial tonsillitis attending at ENT Department of HGH, and who fulflled the inclusion criteria were included consecutively until the sample size was reached. However, due to the lack of suffcient throat swabs and incomplete questionnaires, only 374 children aged from 2 to 5 years suspected of bacterial tonsillitis participated in the study.

Variables.
Bacterial throat culture positivity was the dependent variable while demographic, clinical, and other explanatory variables were independent variables.

Data Collection.
A structured questionnaire was used to collect data on demographic characteristics, clinical profles, and other variables. Data on child's age, mother's age, father's age, gender, residence, maternal and paternal education, parental occupation, type of breast feeding, attending daycare and school, living in overcrowded environments, and exposure to wood biofuels were collected with faceto-face interviews of their caregiver using a structured questionnaire. Moreover, clinical information such as history of contact with someone who had cough, history of tonsillitis, the current type of tonsillitis, number of previous tonsillitis, body temperature, sore throat, swollen tonsils, headache, swollen lymph nodes, difculty in swallowing, white exudates on the throat, weight loss, tonsillar structural change, and history of drug use) were collected by the attending pediatricians.

Troat Swab Sample Collection and Processing.
Troat swabs were taken by the attending pediatricians from each patient using a sterile cotton swab. Visible exudates or hyperemic areas on the tonsillar walls were swabbed with a sterile cotton swab, while the tongue was depressed by a wooden spatula when necessary. All swab samples were immediately transported to the Microbiology Department of HGH using Amie's transport medium (Oxoid, England). Swabs were simultaneously plated onto Tryptic Soy Agar (Himedia, India) containing 5% sheep blood, chocolate agar (CA), and MacConkey (MAC) Agar (Himedia, India) and incubated for 48 h at 37°C. Chocolate agar was incubated in a candle jar to get 5% CO 2 , while BA and MAC were incubated under a normal atmosphere.

Identifcation of Bacterial
Isolates. Pure colonies of the bacterial isolates were identifed to the species level following standard enzymatic and biochemical tests [9]. White to grey large or small colony forming units with a zone of beta hemolysis around 2-3 mm in diameter surrounding each colony plus, Gram positive cocci arranged in a chain and were both coagulase and catalase negative were taken as beta-hemolytic streptococci isolates. Small, shiny, and translucent colonies surrounded by a zone of alpha hemolysis on BA and were Gram positive and susceptible to optochin were identifed as S. pneumoniae isolates. S. aureus isolates were identifed by Gram positive clusters forming glistering golden yellow colonies on BA and mannitol salt agar (MSA) which were coagulase, catalase, and oxidase positive. Moraxella catarrhalis were identifed by nonhemolytic grey to white colonies on blood agar (BA) which were oxidase, and catalase positive. K. pneumoniae and P. aeruginosa isolates were identifed by standard biochemical tests.

Antimicrobial Susceptibility
Testing. Susceptibilities of all identifed bacterial isolates to diferent antimicrobials were performed on Mueller-Hinton agar (MHA) containing 5% sheep's blood (Himedia, India) according to the criteria of the 2019 Clinical and Laboratory Standards Institute (CLSI) (10) using the Kirby-Bauer disk difusion method.
Te following drug discs were tested: ampicillin (10 μg), amoxicillin-clavulanic acid (20/10 μg), cefoxitin (30 μg), gentamicin (10 μg), clarithromycin (15 μg), erythromycin (15 μg), ofoxacin (5 μg), and ciprofoxacin (5 μg). Tese antibiotic discs were selected based on the frequent prescriptions of these drugs for the treatment of tonsillitis infection in the study area and using the CLSI guideline recommendations [10]. A loop full of culture was taken from a pure culture colony and transferred to a tube containing 5 ml of normal saline and mixed gently until it forms a homogenous suspension. Te turbidity of the suspension was then adjusted to the turbidity of McFarland 0.5 (which carries 10 8 CFU/ml) and was swabbed on a dry surface of MHA plate with 5% sheep blood (150 mm) using a sterile cotton swab. Antibiotic discs were dispensed using a single disc dispenser. Plates were then incubated for 24 h at 37°C. Diameters of the zone of inhibition around the discs were measured using a digital caliper. Te results of the zone of inhibition of antibiotics were interpreted based on the 2019 CLSI guideline [11]. All S. aureus isolates were subjected to cefoxitin disc difusion test on Mueller-Hinton agar plates. Plates were incubated at 35°C for 18 h and inhibition zones with a diameter of ≤21 mm were reported as methicillin resistant and ≥22 mm considered as methicillin sensitive. Bacterial isolates that revealed acquired nonsusceptibility to at least one agent in three or more antibiotic categories were considered MDR [10].

Quality
Control. Data collectors were trained on the aim of the study and data collection procedures. Te completeness of data was also checked. Te proper functioning of materials, equipment, culture media, and procedures were checked. Specimens were collected following standard bacteriological procedures. To prevent contamination, all throat swabs were analyzed within two hours of collection. Culture media were checked for sterility by incubating 5% of each batch of the medium at 37°C for 24 hrs. Te performance of all prepared culture media was checked by inoculating with the American Type Culture Collection (ATCC) standard reference strains S. aureus (ATCC 29213), S. penumoniae (ATCC 49618), and P. aeruginosa (ATCC 27853).

Data
Analysis. Data were entered and analyzed using SPSS version 25 (IBM Corp, Armonk, NY, USA). Univariate analysis was made to generate summary values for the most important variables. Logistic regression analysis was made to determine the association between dependent and independent variables. Te generated data were compiled with frequency tables and other statistical summary measures. A stepwise logistic regression model was used to fnd factors associated with culture positive bacterial tonsillitis and statistical signifcance was set at P < 0.05.

Characteristics of the Study Participants.
A total of 374 children aged 2-5 years suspicion of bacterial tonsillitis participated in the study, making a response rate of 97.4% (374/384). Among them, 200 (53.5%) were males. Most (305, 81.6%) of the children were urban residents. Te age range of the children was 2 to 5 years. Te majority (141, 37.7%) of the children were fve years old. Te age of children's mother ranged from 20 to 45 years. Most (69%) of the parents were employees (Table 1).

Isolation Rate of Bacterial Troat Culture.
Overall, 120 (32.1%) of the children were positive for bacterial throat culture. Te proportion of throat culture positivity was higher in males (73, 36.5%) than in females (47, 27%). It was higher in urban (101, 33.1%) than rural (19,27.5%) residents. Te percentage of throat culture positivity was higher (78, 41.9%) in children from mothers who are unable to read and write than in the other groups (5.9-26.7%). Children from fathers who had higher educational attainment had the lowest percentage of throat culture positivity compared to others (Table 1). Table 2 depicts the results of bacterial throat culture with clinical profles among children 2-5 years of age. Most children had presented with acute tonsillitis 202 (54%) and sore throat (343, 91.7%). Swollen tonsils were presented in 367 (98.1%) of children. On the other hand, swollen lymph nodes were presented in 151 (40.4%) children. Moreover, 147 (39.3%) and 69 (18.4%) of the children had difculty swallowing and white exudates, respectively ( Table 2).

Bacterial Troat Culture and Clinical Profles.
Of the total, 172 (46%) of the children were positive for bacterial throat culture. Te percentage of positive bacterial throat cultures was higher among children with a history of tonsillitis 95 (55.2%) than the others 25 (12.4%). Te percentage of throat culture positivity was the highest (30, 57.7%) in children with symptoms of chronic tonsillitis. Children with swollen tonsils had a higher percentage of throat culture positivity rates (119, 32.4%) than those without swollen tonsils (1, 14.3%). Te proportion of throat culture isolation was higher among children who had weight loss (67, 38.2%) than their counterparts (53, 61.8%) ( Table 2).

Bacterial Troat Culture in Relation to Other Variables.
Overall, 96 (25.7%) and 228 (61%) of the children were exclusively breastfed and had a history of contact with coughing patients, respectively. On the other hand, 86 (23%) and 282 (75.4%) of the children were daycare center attendees and school attendees, respectively. Most of the children lived in a crowded houses (268, 71.7%) and 331 (88.5%) had exposure to biofuels (Table 3). Te percentage of positive bacterial throat cultures was higher among children who had a history of contact with coughing patients (85, 37.3%) than their counterparts (35, 24%). Daycare center attendee children had a higher (34/86, 39.5%) percentage of bacterial isolation than others 86 (29.9%). Moreover, school-attending children had a higher (48,52.2%) percentage of bacterial isolation than their counterparts (72, 25.5%). Te proportion of bacterial isolation was higher among children who had exposure to biofuels (117, 35.3%) than their counterparts (3, 7%) ( Table 3). International Journal of Microbiology 5

Multiple Drug Resistant (MDR) Profles of Bacterial
Isolates. Multidrug resistance (MDR) is the resistance of a bacterial isolate to three or more antibiotics taken from diferent categories. Overall, 71 (49.7%) of the bacterial species were MDR and 52.6% of beta-hemolytic streptococci were MDR. Te MDR profle of S. aureus and S. pneumoniae isolates were 17 (40.5%) and 6 (60%), respectively (Table 6).

Multivariable Analysis.
Based on multivariable analysis, positivity for bacterial throat culture was signifcantly associated with difculty in swallowing (AOR � 6.99, CI � 3.56-13.13), weight loss (AOR � 0.33, CI � 0.186-0.597), attending school (AOR � 2.98, CI � 1.64-5.42), history of tonsillitis (AOR � 0.12, CI � 0.06-0.21), and exposure to biofuel (AOR � 0.19, CI � 0.04-0.84). Children who had    International Journal of Microbiology difculty swallowing were 7 times more likely to become positive for bacterial throat culture, compared to children who did not have difculty swallowing. Likewise, schoolattending children were 3 times more likely to be positive for bacterial throat culture compared to nonattenders. Children with a history of tonsillitis were more likely to have a positive bacterial throat culture than those without a history of tonsillitis. Similarly, children who had weight loss and exposure to biofuels were more likely to become positive for throat culture compared to those who did not have weight loss and exposure to biofuels (Table 7).

Discussion
Tonsillitis has a considerably negative impact on the patients' quality of life and has a signifcant burden on public health. Untreated childhood tonsillitis leads to peritonsillar abscess, tonsillar stones, and rheumatic fever. Terefore, identifcation of bacterial isolates and determination of antibiotic susceptibility profles from throat a swab of children with suspicion of bacterial tonsillitis is useful for the treatment of tonsillitis in the healthcare setting of Somaliland where patients are treated without a routine culture diagnosis. Terefore, this study presents the frst report of the antibiotic resistance profles of bacterial isolates from throat swab cultures of children with suspicion of bacterial tonsillitis in HGH. In this study, 32.1% of children 2-5 years of age were positive for bacterial throat culture. Due to lack of previously published data in Somaliland, a comparison of countrywide results was not possible. However, the prevailing magnitude of bacterial throat swab culture is higher than similar studies with a prevalence of 11.3% in Ethiopia [12], 20.6% in Tanzania [13], 21.6% in Norway [14], and 19% in Bangladesh [15]. Te prevalence of positive bacterial throat swab culture in this study was lower than studies done in the United Kingdom (79%) [16], Trinidad (62.5%) [17], India (72%) [18], Saudi Arabia (65%) [19], Benin (73.97%) [20], and Ethiopia (51%) [21]. Te lower rate of positive bacterial throat culture in the present study compared to other developing countries might be attributed to diferences in geography, community living status and hygienic practices, host factor, and educational level of the parents.
Te proportion of positive bacterial throat swab culture in children 2-5 years of age was higher in males than in females, which is similar to studies from India [2] and Nigeria [22]. Moreover, the percentage of positive bacterial throat cultures was higher among children living in urban than rural areas in the present study. Tis was similar to studies done in India [2] and Ethiopia [21]. Tis might be due to variation in encountering infected or colonized people, exposure to air pollution from biofuel use, schooling, and house crowding.
In the present study, Staphylococcus aureus was the second most frequent isolate of throat swab cultures from children with suspicion of bacterial tonsillitis with a rate of 29%. Tis could be due to the persistence of S. aureus in the tonsillar tissues, treatment with antimicrobials, and antibiotic resistance. Moreover, S. aureus has the potential to form bioflm which results in recurrent and chronic infection as well as treatment failure. Te isolation of S. aureus as the main agent of tonsillitis has been reported by several authors in Ethiopia [19], Brazil (40%) [31], Trinidad (68.9%) [17], and Nigeria (32.1%) [22].
Te resistance of the isolates to ampicillin was 91.6% and 14.7% for the association of amoxicillin and clavulanate. Te higher resistance to ampicillin by all bacterial isolates might be due to the production of beta-lactamase enzyme, as well as abuse and excessive use of cheap drugs, which can be aforded and administered without culture diagnostic guidance. Tis is a major concern that limits the use of this common therapeutic option in clinical practice in developing countries. Te rate of ampicillin resistance is comparable to reports from Nigeria (100%) [20,52].
Although the existence of anginosus streptococci group is more likely, the resistance rate of 94.9% of beta-hemolytic streptococci to ampicillin is worrisome as B-lactam antibiotics are the drug of choice for strep throat. Terefore, further study on the molecular characterization of species of Streptococcus from children with throat swab culture is recommended. Te percentage of beta-hemolytic  [53]. Tis likely is due to the enzymatic inactivation mediated by aminoglycoside-modifying enzymes (AMEs) and point mutations in the quinolone resistance-determining region (QRDR).
One of the major worries when determining the resistance profles of isolates is the availability of MDR strains. In this study, half of the bacterial isolates were MDR. Tis is a serious problem for children 2-5 years of age in Somaliland. Children involved in the study area were outpatients and they might have constant contact with other children and their families. Moreover, in the study area, there is no routine culture and antimicrobial susceptibility testing and management of children with tonsillitis are empirical. Tese may result from repeated infections of the tonsils, pyogenic meningitis, rheumatic fever, lower respiratory tract infections, and diffculty to select efective antibiotics. Furthermore, the existence of MDR isolates demonstrates the persistence of the bacteria and the possibility of antimicrobial resistance, dissemination, and recurrence of infection [37].
Te percentage of MDR S. pneumoniae (60%) in this study was higher than studies from Poland (52.9%) [58], Lithuania (12.5%) [56], and Vietnam (35%) [59]. In this study, all isolates of Pseudomonas aeruginosa were MDR (100%), which is concurrent with a study in Brazil (100%) [39]. Tese high proportions of MDR among the isolates might be due to productions of beta-lactamase enzyme by Pseudomonas aeruginosa and the production of penicillin binding proteins in Streptococcus pneumoniae.
In the present study, difculty in swallowing is one of the predictors for positive bacterial throat culture in children with suspicion of bacterial tonsillitis. Similar fndings were reported in India [2] and Lithuania (48). History of tonsillitis was also a predictor variable in this study, which was similar to studies done in Ethiopia [53], and Yemen [55]. Tese might be due to cohabitations of the tonsils by multiple bacterial isolates as depicted in Table 4 and failure of penicillin and ampicillin therapies.
Weight loss was also another predictor for bacterial tonsillitis in this study in which similar studies were reported in Iran [54] and Germany [58]. Furthermore, attending school was a risk factor for tonsillitis in this study similar to studies done in Uganda [57]. Tis might be due to overcrowding during schooling among children where carrier children can easily interact with healthy children.

Limitations of the Study.
Tis study provided the frst report of data on the profle of bacterial throat cultures and their resistance to antibiotics from children 2 to 5 years of age with suspicion of bacterial tonsillitis at Hargeisa Group of Hospital. However, the study was limited to identifying nonbacterial causes of tonsillitis. Due to the limited resources, colonizations and causes were not diferentiated and bacterial isolates were not identifed with molecular techniques like PCR and MALDI-TOF MS. Furthermore, bacterial isolates resistant to ampicillin were not further confrmed by minimum inhibitory concentrations (MIC). Terefore, the interpretive conclusions and recommendations of this study should be based on the fndings of isolation and phenotypic identifcation methods.

Conclusions
A high prevalence of positive bacterial throat swab cultures resistant to diferent antibiotics, MRSA, and mixed isolates was found. Beta-hemolytic streptococci followed by S. aureus and S. pneumoniae were the most frequent isolates. Most of the bacterial isolates were resistant to ampicillin. However, amoxicillin-clavulanic acid and ciprofoxacin are the least resistant drugs. Terefore, the result points out that treatment tonsillitis due to bacteria guided by throat swab culture and antimicrobial susceptibility testing. Further investigations to diferentiate colonization and pathogens, identify nonbacterial causes of tonsillitis, diferentiate species of beta-hemolytic, conduct studies covering larger geographical areas to draw the magnitude, and topographic variations are needed to control the spread of tonsillitis among children within fve years of age.

Data Availability
Te data used to support the fndings of this study are included within the article.

Ethical Approval
Tis study was approved by the Institutional Review Board (IRB) of College of Medicine and Health Science (CMHS) with protocol number (001/2020), Bahir Dar University, and a permission letter was obtained from the Ministry of Health, Somaliland, and Hargeisa Group of Hospitals (HGH). Ethical approval protocol number: CMHS/IRB-001/2020.

Consent
Following well-versed about the purpose and importance of the study, written informed consent was obtained from child and parents/guardians before collecting data. Information obtained from the study participants was kept confdential and used only for this study. Bacteriological positive results were submitted to health pediatricians.

Disclosure
A preprint has previously been published [59]. Te funders had no role in the overall design and conduction of the study.

Conflicts of Interest
Te authors declare that there were no conficts of interest regarding the publication of this article.

Authors' Contributions
HHD conceptualized and designed the study, performed the laboratory work, collected, and managed the data, analyzed and interpreted the results, and contributed to the scientifc content of the manuscript. AM designed the study, performed overall coordination, supervised and facilitated the data collection and management, interpreted the results, critically reviewed or edited the manuscript, and signifcantly contributed to the scientifc content of the study. MK interpreted the results, drafted, critically reviewed, edited, and signifcantly contributed to the scientifc content of the manuscript. WM designed the study, performed overall coordination, supervised and facilitated the data collection and management, analyzed data, interpreted the results, drafted the manuscripts, and signifcantly contributed to the scientifc content of the study. All authors read and approved the fnal manuscript.